Nematicidal compositions and methods

ABSTRACT

The invention concerns the use of certain compounds related to fatty acids to control nematodes that infest plants or the situs of plants. Nematodes that parasitize animals can also be controlled using the methods and composition of this invention. Certain of the useful compounds are fatty acid esters that are predicted inhibitors of nematode delta-12 fatty acid desaturases and can be, for example, from C16 to C20 in length.

RELATED APPLICATION INFORMATION

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 10/090,527, filed Mar. 4, 2002, which is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

[0002] Nematodes (derived from the Greek word for thread) are active,flexible, elongate, organisms that live on moist surfaces or in liquidenvironments, including films of water within soil and moist tissueswithin other organisms. While only 20,000 species of nematode have beenidentified, it is estimated that 40,000 to 10 million actually exist.Some species of nematodes have evolved as very successful parasites ofboth plants and animals and are responsible for significant economiclosses in agriculture and livestock and for morbidity and mortality inhumans (Whitehead (1998) Plant Nematode Control. CAB International, NewYork).

[0003] Nematode parasites of plants can inhabit all parts of plants,including roots, developing flower buds, leaves, and stems. Plantparasites are classified on the basis of their feeding habits into thebroad categories, migratory ectoparasites, migratory endoparasites, andsedentary endoparasites. Sedentary endoparasites, which include the rootknot nematodes (Meloidogyne) and cyst nematodes (Globodera andHeterodera) induce feeding sites and establish long-term infectionswithin roots that are often very damaging to crops (Whitehead, supra).It is estimated that parasitic nematodes cost the horticulture andagriculture industries in excess of $78 billion worldwide a year, basedon an estimated average 12% annual loss spread across all major crops.For example, it is estimated that nematodes cause soybean losses ofapproximately $3.2 billion annually worldwide (Barker et al. (1994)Plant and Soil Nematodes: Societal Impact and Focus for the Future. TheCommittee on National Needs and Priorities in Nematology. CooperativeState Research Service, US Department of Agriculture and Society ofNematologists). Several factors make the need for safe and effectivenematode controls urgent. Continuing population growth, famines, andenvironmental degradation have heightened concern for the sustainabilityof agriculture, and new government regulations may prevent or severelyrestrict the use of many available agricultural anthelmintic agents.

[0004] The situation is particularly dire for high value crops such asstrawberries and tomatoes where chemicals have been used extensively tocontrol of soil pests. The soil fumigant methyl bromide has been usedeffectively to reduce nematode infestations in a variety of thesespecialty crops. It is however regulated under the U.N. MontrealProtocol as an ozone-depleting substance and is scheduled forelimination in 2005 in the US (Carter (2001) California Agriculture,55(3):2). It is expected that strawberry and other commodity cropindustries will be significantly impacted if a suitable replacement formethyl bromide is not found. Presently there are a very small array ofchemicals available to control nematodes and they are frequentlyinadequate, unsuitable, or too costly for some crops or soils (Becker(1999) Agricultural Research Magazine 47(3):22-24; U.S. Pat. No.6,048,714). The few available broad-spectrum nematicides such as Telone(1,3-dichloropropene+chloropicrin) have significant restrictions ontheir use because of toxicological concerns (Carter (2001) CaliforniaAgriculture 55(3): 12-18).

[0005] Fatty acids are a class of natural compounds that have beeninvestigated as alternatives to the toxic, non-specific organophosphate,carbamate and fumigant pesticides (Stadler et al. (1994) Planta Medica60(2):128-132; U.S. Pat. Nos. 5,192,546; 5,346,698; 5,674,897;5,698,592; 6,124,359). It has been suggested that fatty acids derivetheir pesticidal effects by adversely interfering with the nematodecuticle or hypodermis via a detergent (solubilization) effect, orthrough direct interaction of the fatty acids and the lipophilic regionsof target plasma membranes (Davis et al. (1997) Journal of Nematology29(4S):677-684). In view of this general mode of action it is notsurprising that fatty acids are used in a variety of pesticidalapplications including as herbicides (e.g., SCYTHE by Dow Agrosciencesis the C9 saturated fatty acid pelargonic acid), as bacteriacides andfungicides (U.S. Pat. Nos. 4,771,571; 5,246,716) and as insecticides(e.g., SAFER INSECTICIDAL SOAP by Safer, Inc.).

[0006] The phytotoxicity of fatty acids has been a major constraint ontheir general use in agricultural applications (U.S. Pat. No. 5,093,124)and the mitigation of these undesirable effects while preservingpesticidal activity is a major area of research. The esterification offatty acids can significantly decrease their phytotoxicity (U.S. Pat.Nos. 5,674,897; 5,698,592; 6,124,359). Such modifications can howeverlead to dramatic loss of nematicidal activity as is seen for linoleic,linolenic and oleic acid (Stadler et al. (1994) Planta Medica60(2):128-132) and it may be impossible to completely decouple thephytotoxicity and nematicidal activity of pesticidal fatty acids becauseof their non-specific mode of action. Perhaps not surprisingly, thenematicidal fatty acid pelargonic acid methyl ester (U.S. Pat. Nos.5,674,897; 5,698,592; 6,124,359) shows a relatively small “therapeuticwindow” between the onset of pesticidal activity and the observation ofsignificant phytotoxicity (Davis et al. (1997) J Nematol29(4S):677-684). This is the expected result if both the phytotoxicityand the nematicidial activity derive from the non-specific disruption ofplasma membrane integrity. Similarly the rapid onset of pesticidalactivity seen with many nematicidal fatty acids at therapeuticconcentrations (U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359) suggestsa non-specific mechanism of action, possibly related to the disruptionof membranes, action potentials and neuronal activity.

[0007] Ricinoleic acid, the major component of castor oil, providesanother example of the unexpected effects esterification can have onfatty acid activity. Ricinoleic acid has been shown to have aninhibitory effect on water and electrolyte absorption using evertedhamster jejunal and ileal segments (Gaginella et al. (1975) J PharmacolExp Ther 195(2):355-61) and to be cytotoxic to isolated intestinalepithelial cells (Gaginella et al. (1977) J Pharmacol Exp Ther201(1):259-66). These features are likely the source of the laxativeproperties of castor oil which is given as a purgative in humans andlivestock (e.g., castor oil is a component of some de-worming protocolsbecause of its laxative properties). In contrast, the methyl ester ofricinoleic acid is ineffective at suppressing water absorption in thehamster model (Gaginella et al. (1975) J Pharmacol Exp Ther195(2):355-61).

[0008] The macrocyclic lactones (e.g., avermectins and milbemycins) anddelta-toxins from Bacillus thuringiensis (Bt) are chemicals that inprinciple provide excellent specificity and efficacy and should allowenvironmentally safe control of plant parasitic nematodes.Unfortunately, in practice, these two approaches have proven lesseffective for agricultural applications against root pathogens. Althoughcertain avermectins show exquisite activity against plant parasiticnematodes these chemicals are hampered by poor bioavailability due totheir light sensitivity, degradation by soil microorganisms and tightbinding to soil particles (Lasota & Dybas (1990) Acta Leidlen59(1-2):217-225; Wright & Perry (1998) Musculature and Neurobiology. In:The Physiology and Biochemistry of Free-Living and Plant-parasiticNematodes (eds. Perry & Wright), CAB International 1998). Consequentlydespite years of research and extensive use against animal parasiticnematodes, mites and insects (plant and animal applications),macrocyclic lactones (e.g., avermectins and milbemycins) have never beencommercially developed to control plant parasitic nematodes in the soil.

[0009] Bt toxins must be ingested to affect their target organ the brushborder of midgut epithelial cells (Marroquin et al. (2000) Genetics.155(4):1693-1699). Consequently they are not anticipated to be effectiveagainst the dispersal, non-feeding, juvenile stages of plant parasiticnematodes in the field. These juvenile stages only commence feeding whena susceptible host has been infected, thus effective nematicides must beable to penetrate the cuticle. In addition, soil mobility of arelatively large 65-130 kDa protein—the size of typical Bt deltatoxins—is expected to be poor and delivery in planta is likely to beconstrained by the exclusion of large particles by the feeding tube ofcertain plant parasitic nematodes such as Heterodera (Atkinson et al.(1998) Engineering resistance to plant-parasitic nematodes. In: ThePhysiology and Biochemistry of Free-Living and Plant-parasitic Nematodes(eds Perry & Wright), CAB International 1998).

[0010] Many plant species are known to be highly resistant to nematodes.The best documented of these include marigolds (Tagetes spp.), rattlebox(Crotalaria spectabilis), chrysanthemums (Chrysanthemum spp.), castorbean (Ricinus communis), margosa (Azardiracta indica), and many membersof the family Asteraceae (family Compositae) (Hackney & Dickerson.(1975) J Nematol 7(1):84-90). The active principle(s) for thisnematicidal activity has not been discovered in all of these examplesand no plant-derived products are sold commercially for control ofnematodes. In the case of the Asteraceae, the photodynamic compoundalpha-terthienyl has been shown to account for the strong nematicidalactivity of the roots. In the case of castor beans they are plowed underas a green manure before a seed crop is set. A significant drawback ofthe castor plant is that the seed contains toxic compounds (such asricin) that can kill humans, pets, and livestock and is also highlyallergenic.

[0011] There remains an urgent need to develop environmentally safe,target-specific ways of controlling plant parasitic nematodes. In thespecialty crop markets, economic hardship resulting from nematodeinfestation is highest in strawberries, bananas, and other high valuevegetables and fruits. In the high-acreage crop markets, nematode damageis greatest in soybeans and cotton. There are however, dozens ofadditional crops that suffer from nematode infestation including potato,pepper, onion, citrus, coffee, sugarcane, greenhouse ornamentals andgolf course turf grasses.

[0012] Nematode parasites of vertebrates (e.g., humans, livestock andcompanion animals) include gut roundworms, hookworms, pinworms,whipworms, and filarial worms. They can be transmitted in a variety ofways, including by water contamination, skin penetration, bitinginsects, or by ingestion of contaminated food.

[0013] In domesticated animals, nematode control or “de-worming” isessential to the economic viability of livestock producers and is anecessary part of veterinary care of companion animals. Parasiticnematodes cause mortality in animals (e.g., heartworm in dogs and cats)and morbidity as a result of the parasites' inhibiting the ability ofthe infected animal to absorb nutrients. The parasite-induced nutrientdeficiency results in diseased livestock and companion animals (i.e.,pets), as well as in stunted growth. For instance, in cattle and dairyherds, a single untreated infection with the brown stomach worm canpermanently stunt an animal's ability to effectively convert feed intomuscle mass or milk.

[0014] Two factors contribute to the need for novel anthelmintics andvaccines for control of parasitic nematodes of animals. First, some ofthe more prevalent species of parasitic nematodes of livestock arebuilding resistance to the currently available anthelmintic drugs,meaning that these products will eventually lose their efficacy. Thesedevelopments are not surprising because few effective anthelmintic drugsare available and most have been used continuously. Presently a numberof parasitic species has developed resistance to most of theanthelmintics (Geents et al. (1997) Parasitology Today 13:149-151;Prichard (1994) Veterinary Parasitology 54:259-268). The fact that manyof the anthelmintic drugs have similar modes of action complicatesmatters, as the loss of sensitivity of the parasite to one drug is oftenaccompanied by side resistance - that is, resistance to other drugs inthe same class (Sangster & Gill (1999) Parasitology Today15(4):141-146). Secondly, there are some issues with toxicity for themajor compounds currently available.

[0015] Human infections by nematodes result in significant mortality andmorbidity, especially in tropical regions of Africa, Asia, and theAmericas. The World Health Organization estimates 2.9 billion people areinfected with parasitic nematodes. While mortality is rare in proportionto total infections (180,000 deaths annually), morbidity is tremendousand rivals tuberculosis and malaria in disability adjusted life yearmeasurements. Examples of human parasitic nematodes include hookworm,filarial worms, and pinworms. Hookworm is the major cause of anemia inmillions of children, resulting in growth retardation and impairedcognitive development. Filarial worm species invade the lymphatics,resulting in permanently swollen and deformed limbs (elephantiasis) andinvade the eyes causing African Riverblindness. Ascaris lumbricoides,the large gut roundworm infects more than one billion people worldwideand causes malnutrition and obstructive bowl disease. In developedcountries, pinworms are common and often transmitted through children indaycare.

[0016] Even in asymptomatic parasitic infections, nematodes can stilldeprive the host of valuable nutrients and increase the ability of otherorganisms to establish secondary infections. In some cases, infectionscan cause debilitating illnesses and can result in anemia, diarrhea,dehydration, loss of appetite, or death.

[0017] While public health measures have nearly eliminated one tropicalnematode (the water-borne Guinea worm), cases of other worm infectionshave actually increased in recent decades. In these cases, drugintervention provided through foreign donation or purchased by those whocan afford it remains the major means of control. Because of the highrates of reinfection after drug therapy, vaccines remain the best hopefor worm control in humans. There are currently no vaccines available.

[0018] Until safe and effective vaccines are discovered to preventparasitic nematode infections, anthelmintic drugs will continue to beused to control and treat nematode parasitic infections in both humansand domestic animals. Finding effective compounds against parasiticnematodes has been complicated by the fact that the parasites have notbeen amenable to culturing in the laboratory. Parasitic nematodes areoften obligate parasites (i.e., they can only survive in theirrespective hosts, such as in plants, animals, and/or humans) with slowgeneration times. Thus, they are difficult to grow under artificialconditions, making genetic and molecular experimentation difficult orimpossible. To circumvent these limitations, scientists have usedCaenorhabidits elegans as a model system for parasitic nematodediscovery efforts.

[0019]C. elegans is a small free-living bacteriovorous nematode that formany years has served as an important model system for multicellularanimals (Burglin (1998) Int. J. Parasitol. 28(3): 395-411). The genomeof C. elegans has been completely sequenced and the nematode shares manygeneral developmental and basic cellular processes with vertebrates(Ruvkin et al. (1998) Science 282: 2033-41). This, together with itsshort generation time and ease of culturing, has made it a model systemof choice for higher eukaryotes (Aboobaker et al. (2000) Ann. Med. 32:23-30).

[0020] Although C. elegans serves as a good model system forvertebrates, it is an even better model for study of parasiticnematodes, as C. elegans and other nematodes share unique biologicalprocesses not found in vertebrates. For example, unlike vertebrates,nematodes produce and use chitin, have gap junctions comprised ofinnexin rather than connexin and contain glutamate-gated chloridechannels rather than glycine-gated chloride channels (Bargmann (1998)Science 282: 2028-33). The latter property may be of particularrelevance given that the avennectin class of drugs is thought to act atglutamate-gated chloride receptors and is highly selective forinvertebrates (Martin (1997) Vet. J. 154:11-34).

[0021] A subset of the genes involved in nematode specific processeswill be conserved in nematodes and absent or significantly diverged fromhomologues in other phyla. In other words, it is expected that at leastsome of the genes associated with functions unique to nematodes willhave restricted phylogenetic distributions. The completion of the C.elegans genome project and the growing database of expressed sequencetags (ESTs) from numerous nematodes facilitate identification of these“nematode specific” genes. In addition, conserved genes involved innematode-specific processes are expected to retain the same or verysimilar functions in different nematodes. This functional equivalencehas been demonstrated in some cases by transforming C. elegans withhomologous genes from other nematodes (Kwa et al. (1995) J. Mol. Biol.246:500-10; Redmond et al. (2001) Mol. Biochem. Parasitol. 112:125-131).This sort of data transfer has been shown in cross phyla comparisons forconserved genes and is expected to be more robust among species within aphylum. Consequently, C. elegans and other free-living nematode speciesare likely excellent surrogates for parasitic nematodes with respect toconserved nematode processes.

[0022] Many expressed genes in C. elegans and certain genes in otherfree-living nematodes can be “knocked out” genetically by a processreferred to as RNA interference (RNAi), a technique that provides apowerful experimental tool for the study of gene function in nematodes(Fire et al. (1998) Nature 391(6669):806-811; Montgomery et al. (1998)Proc. Natl. Acad Sci USA 95(26):15502-15507). Treatment of a nematodewith double-stranded RNA of a selected gene can destroy expressedsequences corresponding to the selected gene thus reducing expression ofthe corresponding protein. By preventing the translation of specificproteins, their functional significance and essentiality to the nematodecan be assessed. Determination of essential genes and theircorresponding proteins using C. elegans as a model system will assist inthe rational design of anti-parasitic nematode control products.

[0023] The present invention describes compositions which showsurprising nematicidal activity in part due to selective inhibition ofmetabolic processes demonstrated to be essential to nematodes and eitherabsent or non-essential in vertebrates and plants. This inventiontherefore provides urgently needed compounds and methods for theenvironmentally safe control of parasitic nematodes.

SUMMARY

[0024] The invention concerns compositions and processes for controllingnematodes. In one embodiment, the subject invention comprises the use ofcertain compounds related to fatty acids to control nematodes thatinfest plants or the situs of plants. Nematodes that parasitize animalscan also be controlled using the methods and compounds of thisinvention.

[0025] Certain of the useful nematicidal fatty acid analogs arepredicted inhibitors of nematode delta-12 fatty acid desaturases (alsoreferred to herein as a nematode delta-12 desaturases). The useful fattyacid analogs can be, for example, an epoxide, a cyclopropane, acyclopropene, methylated, an oxo, or hydroxylated analog. The analogscan also contain sulfur in place of carbon at certain positions. In apreferred embodiment of the subject invention the fatty acid analog is adelta-12 desaturase inhibiting fatty acid ester.

[0026] Preferred fatty acid esters useful according to the subjectinvention are C16 to C20 in length, have a cis (Z) or a trans (E) carbondouble bond at the delta-9 position (i.e., between C9 and C10 countingfrom the carbonyl carbon (C═O)) and a variety of modifications at theC12, C13 or both C12 and C13 positions. Preferred fatty acid esters alsoinclude thia fatty acid esters with sulfur in place of carbon atpositions 12, 13 or 12 and 13. Most preferred compounds are C16 to C18in length. Examples include, ricinoleic acid methyl ester(12-hydroxy-cis-9-octadecenoic acid methyl ester), ricinelaidic acidmethyl ester (12-hydroxy-trans-9-octadecenoic acid methyl ester),vernolic acid methyl ester ((12,13)-epoxy-cis-9-octadecenoic acid methylester), 12-oxo-9(Z)-octadecenoic acid methyl ester and crepenynic acidmethyl ester (9(Z)-octadecen-12-ynoic acid methyl ester). Specificallyexcluded are the normal substrates of delta-12 desaturases (e.g.,cis-9-octadecenoate (oleate), cis-9-hexadecenoate (palmitoleate),isomers of the substrate such as trans-9-octadecenoate (elaidate) andthe normal products of delta-12 desaturases (e.g.,cis-9,12-octadecadienoate (linoleate), cis-9,12-hexadecadienoate). Fattyacid compounds where the only modifications are a single cis or transdouble bond at the delta-9 position (i.e., a cis or trans double bondbetween C9 and C10), or double bonds at both the delta-9 (cis or transdouble bond between C9 and C10) and delta-12 positions (i.e., a cis ortrans double bond between C12 and C13) as well as certain naturallyoccurring esters such as triglycerides, diacylglycerides andphospholipids are generally not preferred. Examples of preferred sulfurcontaining fatty acid analogs include methyl 12-thia-oleate and methyl13-thia-oleate.

[0027] Fatty acid analogs that have the characteristics of a specificinhibitor of delta-12 desaturase inhibit the activity of a nematodedelta-12 desaturase to a lesser extent in the presence of the product ofa delta-12 fatty acid desaturase (e.g., linoleate) than in the presenceof the substrate of the enzyme (e.g., oleate). For these competitionexperiments the substrate (e.g., oleate) and the product (e.g.,linoleate) are used in equivalent amounts. These effects can bedemonstrated on a delta-12 fatty acid desaturase (also referred toherein as a delta-12 desaturase) protein in vitro, on transgenic cellscontaining delta-12 desaturases or on intact organisms (e.g., anematode) containing delta-12 desaturases. In one embodiment of thistest, the inhibitor, the substrate and product of the delta-12desaturase are present in equal concentrations.

[0028] The invention also features compounds that inhibit the expressionof a delta-12 desaturase at the level of transcription or translation.Also within the invention are compounds that that impair themodification of a delta-12 desaturase resulting in change in theactivity or localization of the desaturase.

[0029] The invention also features compounds that are relativelyselective inhibitors of one or more nematode delta-12 desaturasepolypeptides relative to one or more plant or animal fatty aciddesaturase-like polypeptides. The compounds can have a K₁ for a nematodefatty acid desaturase that is 10-fold, 100-fold, 1,000-fold or morelower than for a plant or animal fatty acid desaturase-likepolypeptides, e.g., a host plant or host animal of the nematode. Theinvention further features relatively non-selective inhibitors as wellas completely non-selective inhibitors.

[0030] In yet another aspect, the invention features a method oftreating a disorder (e.g., an infection) caused by a nematode, (e.g., M.incognita, H. glycines, H. contortus, A. suum) in a subject, e.g., ahost plant or host animal. The method includes administering to thesubject an effective amount of a compound of the invention, e.g., aninhibitor of a delta-12 desaturase polypeptide activity or an inhibitorof expression of a delta-12 desaturase polypeptide or an inhibitor thatimpairs the modification of a delta-12 desaturase resulting in change inthe activity or localization of the desaturase. The inhibitor may bedelivered by several means including as a feed additive, as a pill or byinjection.

[0031] In still another aspect, methods of inhibiting a nematode (e.g.,M. incognita, H. glycines, H. contortus, A. suum) delta-12 desaturase(s)are provided. Such methods can include the steps of: (a) providing anematode that contains a delta-12 fatty acid desaturase-like gene; (b)contacting the nematode with fatty acid analogs or other compounds thatinhibit the enzyme. Also provided are methods of rescuing the effect ofthe inhibitor. Such methods comprise the steps of: (a) inhibiting theenzyme and (b) providing delta-12 unsaturated fatty acids exogenously(e.g., linoleate).

[0032] In another aspect, methods of reducing the viability or fecundityor slowing the growth or development or inhibiting the infectivity of anematode using a nematicidal fatty acid analog of the invention, e.g.,an inhibitor of a delta-12 desaturase are provided. Such methodscomprise the steps of (a) providing a nematode that contains a delta-12desaturase-like gene; (b) contacting the nematode with specific fattyacid analogs, e.g., an inhibitor of a delta-12 fatty acid desaturase;(c) reducing the viability or fecundity of the nematode. Also providedare methods of rescuing the effect of the fatty acid desaturaseinhibitors or other inhibitors. Such methods can involve contacting thenematode with delta-12 unsaturated fatty acids exogenously.

[0033] The invention features a method for reducing the viability,growth, or fecundity of a nematode, the method comprising exposing thenematode to a fatty acid analog of the invention, e.g., a compound thatinhibits the activity of a fatty acid desaturase-like polypeptide (e.g.,a delta-12 fatty acid desaturase) and a method for protecting a plantfrom a nematode infection, the method comprising applying to the plant,to the soil, or to seeds of the plant a fatty acid analog of theinvention. The invention also features a method for protecting a mammalfrom a nematode infection, the method comprising administering to themammal a fatty acid analog of the invention, e.g., an inhibitor of anematode fatty acid desaturase-like polypeptide (e.g., a delta-12 fattyacid desaturase). In preferred embodiments the inhibitor does notsignificantly inhibit the activity of a fatty acid desaturase-likepolypeptide expressed by the plant or at least does not do so to theextent that the growth of the plant is significantly impaired.

[0034] The invention process is particularly valuable to controlnematodes attacking the roots of desired crop plants, ornamental plants,and turf grasses. The desired crop plants can be, for example, soybeans,cotton, strawberries, tomatoes, banana, sugar cane, sugar beet,potatoes, or citrus.

[0035] Thus, the invention features a composition, e.g., a nematicidalcomposition, comprising: (a) an effective amount of a compound havingthe formula:

[0036] wherein: R₁=a C₁-C₅ singly or multiply substituted orunsubstituted carbon chain, wherein the substituants are selected fromthe group consisting of: hydroxy, oxo, halogen, amino, cyano, a singlyor multiply substituted or unsubstituted C₁-C₂ carbon chain,cyclopropane, and epoxy; and R₂=a C₁₅-C₁₉ singly or multiply substitutedor unsubstituted carbon chain having a cis or trans double bond betweenthe 9^(th) and 10^(th) carbons counting form the carbonyl carbon andeither: (i) a triple bond between the 12^(th) and 13^(th) carbonscounting from the carbonyl carbon or (ii) either a single bond or a cisor trans double bond between the 12^(th) and 13^(th) carbons countingfrom the carbonyl carbon and at least one substituant at one or both ofthe 12^(th) and 13^(th) carbons counting from the carbonyl carbon,wherein the substituants are selected from the group consisting ofhydroxy, oxo, halogen, amino, cyano, azido, a singly or multiplysubstituted or unsubstituted C₁-C₂ carbon chain, cyclopropane,cyclopropene, and epoxy; and (b) an aqueous surfactant.

[0037] In certain embodiments, R₁=a C₁-C₅ singly or multiply substitutedor unsubstituted carbon chain, wherein the substituants are selectedfrom the group consisting of: hydroxy, oxo, halogen, amino, cyano, anunsubstituted C₁-C₂ carbon chain, cyclopropane, and epoxy. In otherembodiments, R₂=a C₁₅-C₁₉ singly or multiply substituted orunsubstituted carbon chain having a cis or trans double bond between the9^(th) and 10^(th) carbons counting form the carbonyl carbon and either:(i) a triple bond between the 12^(th) and 13^(th) carbons counting fromthe carbonyl carbon or (ii) either a single bond or a cis or a transdouble bond between the ₁₂ ^(th) and 13^(th) carbons counting from thecarbonyl carbon and at least one substituant at one or both of the12^(th) and 13^(th) carbons counting from the carbonyl carbon, whereinthe substituants are selected from the group consisting of hydroxy, oxo,halogen, amino, cyano, azido, a unsubstituted C₁-C₂ carbon chain,cyclopropane, cyclopropene, and epoxy; the C1-C2 carbon chain of one orboth of R₁ and R₂ is singly or multiple substituted and the substituantsare selected from the group consisting of: hydroxy, oxo, halogen, amino,cyano, and epoxy. In certain embodiments: the C₁-C₂ carbon chain of oneor both of R₁ and R₂ is singly or multiple substituted and thesubstituants are selected from the group consisting of: hydroxy, oxo,halogen, and amino; the C₁-C₂ carbon chain of R₁ is singly substituted;the C₁-C₂ carbon chain of R₂ is singly substituted; and R₁=a singly ormultiply substituted C1 carbon chain R₁ is a C₁-C₂ substituted orunsubstituted carbon chain.

[0038] In other embodiments, R₂ is substituted only at one or both of12^(th) and 13^(th) carbons counting from the carbonyl carbon; R₂ issubstituted only at the 12^(th) carbon counting from the carbonylcarbon; R₂ is substituted only at the 13^(th) carbon counting from thecarbonyl carbon; wherein within R₂ there is a triple bond between the12^(th) and 13^(th) carbons counting from the carbonyl carbon; andwithin R₂ the substituants are selected from the group consisting of:hydroxy, oxo, epoxy, and a C₁ methyl.

[0039] The invention also features a nematicidal composition comprising:(a) a fatty acid methyl ester selected from the group consisting of:ricinoleic acid methyl ester, ricinelaidic acid methyl ester,12-oxo-9(Z)-octadecenoic acid methyl ester, crepenynic acid methylester, and vernolic acid methyl ester; and (b) an aqueous surfactant. Incertain embodiments, the aqueous surfactant is selected from the groupconsisting of: ethyl lactate, Tween 20 and Igepal CO 630; thecomposition comprises a permeation enhancer, e.g., a cyclodextrin. Apermeation enhancer is generally an agent that permits the activecompounds the invention, e.g., the fatty acid esters of the invention,to pass through cellular membranes.

[0040] The composition can further include a co-solvent, e.g.,isopropanol. A co-solvent (i.e., a latent solvent or indirect solvent)is an agent that becomes an effective solvent in the presence of anactive solvent.

[0041] The compositions can also include one more nematicides such as anavermectin, ivermectin, and milbemycin.

[0042] The invention features methods for controlling nematodes byadministering a fatty acid ester of the invention, e.g., a delta-12fatty acid desaturase inhibitor. Thus, the invention includes a methodfor control of unwanted nematodes, the method comprising administeringto mammals, plants, seeds or soil a nematicidal composition comprising:(a) an effective amount of a compound having the formula

[0043] wherein: R₁=a C₁-C₅ singly or multiply substituted orunsubstituted carbon chain, wherein the substituants are selected fromthe group consisting of: hydroxy, oxo, halogen, amino, cyano, a singlyor multiply substituted or unsubstituted C₁-C₂ carbon chain,cyclopropane, and epoxy; and R₂=a C₁₅-C₁₉ singly or multiply substitutedor unsubstituted carbon chain having a cis or trans double bond betweenthe 9^(th) and 10^(th) carbons counting form the carbonyl carbon andeither: (i) a triple bond between the 12^(th) and 13^(th) carbonscounting from the carbonyl carbon or (ii) either a single bond or a cisor a trans double bond between the 12^(th) and 13^(th) carbons countingfrom the carbonyl carbon and at least one substituant at one or both ofthe 12^(th and) 13^(th) carbons counting from the carbonyl carbon,wherein the substituants are selected from the group consisting ofhydroxy, oxo, halogen, amino, cyano, azido, a singly or multiplysubstituted or unsubstituted C₁-C₂ carbon chain, cyclopropane,cyclopropene, and epoxy; and (b) an aqueous surfactant.

[0044] In certain methods of the invention: R₁=a C₁-C₅ singly ormultiply substituted or unsubstituted carbon chain, wherein thesubstituants are selected from the group consisting of: hydroxy, oxo,halogen, amino, cyano, a unsubstituted C₁-C₂ carbon chain, cyclopropane,and epoxy; R₂=a C₁₅-C₁₉ singly or multiply substituted or unsubstitutedcarbon chain having a cis or trans double bond between the 9^(th) and10^(th) carbons counting form the carbonyl carbon and either: (i) atriple bond between the 12^(th) and 13^(th) carbons counting from thecarbonyl carbon or (ii) either a single bond or a cis or a trans doublebond between the 12^(th) and 13^(th) carbons counting from the carbonylcarbon and at least one substituant at one or both of the 12^(th) and13^(th) carbons counting from the carbonyl carbon, wherein thesubstituants are selected from the group consisting of hydroxy, oxo,halogen, amino, cyano, azido, a unsubstituted C₁-C₂ carbon chain,cyclopropane, cyclopropene, and epoxy; the C₁-C₂ carbon chain of one orboth of R₁ and R₂ is singly or multiple substituted and the substituantsare selected from the group consisting of: hydroxy, oxo, halogen, amino,cyano, and epoxy; the C₁-C₂ carbon chain of R₁ is singly substituted;the C₁-C₂ carbon chain of R₂ is singly substituted; R₁ is a C₁-C₂substituted or unsubstituted carbon chain; R₂ is substituted only at oneor both of 12^(th) and 13^(th) carbons counting from the carbonylcarbon; R₂ is substituted only at the 12^(th) carbon counting from thecarbonyl carbon; R₂ is substituted only at the 13^(th) carbon countingfrom the carbonyl carbon; wherein within R₂ there is a triple bondbetween the 12^(th) and 13^(th) carbons counting from the carbonylcarbon; and within R₂ the substituants are selected from the groupconsisting of: hydroxy, oxo, epoxy, and a C₁ methyl.

[0045] The invention also features a method for control of unwantednematodes comprising administering to mammals, plants, seeds or soil anematicidal composition comprising an effective amount of: (a) a fattyacid methyl ester selected from the group consisting of: ricinoleic acidmethyl ester, ricinelaidic acid methyl ester, 12-oxo-9(Z)-octadecenoicacid methyl ester, crepenynic acid methyl ester, and vernolic acidmethyl ester; and (b) an aqueous surfactant.

[0046] In certain embodiments of the method the aqueous surfactant isselected from the group consisting of: ethyl lactate, Tween 20 andIgepal CO 630; the composition comprises a permeation enhancer (e.g., acyclodextrin); the composition comprises a co-solvent (e.g.,isopropanol); the method includes administering (before, after or inconjunction with the fatty acid analog) a nematicide selected from thegroup consisting of: avermectins, ivermectin, and milbemycin; thenematode infects plants and the nematicidal composition is applied tothe soil or to plants; the nematicidal composition is applied to soilbefore planting; the nematicidal composition is applied to soil afterplanting; the nematicidal composition is applied to soil using a dripsystem; the nematicidal composition is applied to soil using a drenchsystem; the nematicidal composition is applied to plant roots; thenematicidal composition is applied to seeds; the nematode infects amammal; the nematicidal composition is administered to non-human mammal;the nematicidal composition is administered to a human; the nematicidalcomposition is formulated as a drench to be administered to a non-humananimal; the nematicidal composition is formulated as an orallyadministered drug; and the nematicidal composition is formulated as aninjectable drug.

[0047] The invention also features feeds that have been supplemented toinclude one or more of the compounds of the invention, e.g., a delta-12fatty acid desaturase inhibitor. The feeds may also be treated to reducethe amount of delta-12 fatty acid desaturase substrates or products inthe feed. More generally, the feed can be treated to reduce the contentof fatty acids that act to complement the loss of a delta-12 fatty aciddesaturase activity.

[0048] Thus, the invention features a nematicidal feed for a non-humanmammal comprising: (a) an animal feed; (b) an effective amount of anematicidal compound having the formula

[0049] wherein: R₁=a C₁-C₅ substituted or unsubstituted carbon chain,wherein the substituants are selected from the group consisting of:hydroxy, oxo, halogen, amino, cyano, a singly or multiply substituted orunsubstituted C₁-C₂ carbon chain, cyclopropane, and epoxy; and R₂=aC₁₅-C₁₉ substituted or unsubstituted carbon chain having a cis or transdouble bond between the 9^(th) and 10^(th) carbons and either: (i) atriple bond between the 12^(th) and 13^(th) carbons or (ii) either asingle bond or a cis or a trans double bond between the 12^(th) and13^(th) carbons and at least one substituant at one or both of the12^(th) and 13^(th) carbons, wherein the substituants are selected fromthe group consisting of hydroxy, oxo, halogen, amino, cyano, azido, asingly or multiply or unsubstituted C₁-C₂ carbon chain, cyclopropane,cyclopropene, and epoxy; and (c) an aqueous surfactant.

[0050] The feed can be treated to reduce linoleic acid content,linolenic acid content or both and the feed can be treated to reduce oneor both of the gamma linolenic acid content and the alpha linolenic acidcontent have been reduced. The feed can be selected from the groupconsisting of: soy, wheat, corn, sorghum, millet, alfalfa, clover, andrye.

[0051] The invention also features a nematicidal composition comprising:(a) an effective amount of a compound having the formula

[0052] wherein: R₁=a C₁-C₅ substituted or unsubstituted carbon chain,wherein the substituants are selected from the group consisting of:hydroxy, oxo, halogen, amino, cyano, a singly or multiply substituted orunsubstituted C₁-C₂ carbon chain, cyclopropane, and epoxy; R₃=a C₁₁substituted or unsubstituted carbon chain having a cis or trans doublebond between the 9^(th) and 10^(th) carbons counting form the carbonylcarbon, wherein the substituants are selected from the group consistingof hydroxy, oxo, halogen, amino, cyano, a singly or multiply orunsubstituted C₁-C₂ carbon chain, cyclopropane, cyclopropene, and epoxy;R₄=a C₂-C₆ substituted or unsubstituted carbon chain wherein thesubstituants are selected from the group consisting of: hydroxy, oxo,halogen, amino, cyano, a singly or multiply substituted or unsubstitutedC₁-C₂ carbon chain, cyclopropane, and epoxy; X and Y are independently asubstituted or unsubstituted methyl or S, provided at least one or X andY is S and wherein the substituants on the methyl selected from thegroup consisting of: oxo, halogen, hydrogen, amino, and hydroxy; and (b)an aqueous surfactant. In certain embodiments, one of X and Y is CH₂.

[0053] The invention also features a nematicidal composition comprising(a) an effective amount of a compound having the formula

[0054] wherein: R₁=a C₁-C₅ substituted or unsubstituted carbon chain,wherein the substituants are selected from the group consisting of:hydroxy, oxo, halogen, amino, cyano, a singly or multiply substituted orunsubstituted C₁-C₂ carbon chain, cyclopropane, and epoxy; and R₂=aC₁₅-C₁₉ substituted or unsubstituted carbon chain having a single bondbetween the 9^(th) and 10^(th) carbons counting form the carbonyl carbonand either: (i) a triple bond between the 12^(th) and 13^(th) carbons or(ii) either a single bond or a cis or a trans double bond between the12^(th) and 13^(th) carbons and at least one substituant at one or bothof the 12^(th) and 13^(th) carbons, wherein the substituants areselected from the group consisting of hydroxy, oxo, halogen, amino,cyano, azido, a substituted or unsubstituted C₁-C₂ carbon chain,cyclopropane, cyclopropene, and epoxy; and (b) an aqueous surfactant.

[0055] In certain embodiments R₂=a C₁₅-C₁₉ substituted or unsubstitutedcarbon chain having a single bond between the 9^(th) and 10^(th) carbonsand a single bond between the 12^(th) and 13^(th) carbons and at leastone substituant at one or both of the 12^(th) and 13^(th) carbons,wherein the substituants are selected from the group consisting ofhydroxy, oxo, halogen, amino, cyano, azido, a singly or multiply orunsubstituted C₁-C₂ carbon chain, cyclopropane, cyclopropene, and epoxy;the 12^(th) and 13^(th) carbons are substituted with an epoxy group; andthe 12^(th) carbon is substituted with a hydroxy group.

[0056] A “purified polypeptide”, as used herein, refers to a polypeptidethat has been separated from other proteins, lipids, and nucleic acidswith which it is naturally associated. The polypeptide can constitute atleast 10, 20, 50, 70, 80 or 95% by dry weight of the purifiedpreparation.

[0057] An “isolated nucleic acid” is a nucleic acid, the structure ofwhich is not identical to that of any naturally occurring nucleic acid,or to that of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term therefore covers, forexample: (a) a DNA which is part of a naturally occurring genomic DNAmolecule but is not flanked by both of the nucleic acid sequences thatflank that part of the molecule in the genome of the organism in whichit naturally occurs; (b) a nucleic acid incorporated into a vector orinto the genomic DNA of a prokaryote or eukaryote in a manner such thatthe resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, or(iii) cell clones in a DNA library such as a cDNA or genomic DNAlibrary. Isolated nucleic acid molecules according to the presentinvention further include molecules produced synthetically, as well asany nucleic acids that have been altered chemically and/or that havemodified backbones.

[0058] Although the phrase “nucleic acid molecule” primarily refers tothe physical nucleic acid molecule and the phrase “nucleic acidsequence” refers to the sequence of the nucleotides in the nucleic acidmolecule, the two phrases can be used interchangeably.

[0059] The term “substantially pure” as used herein in reference to agiven polypeptide means that the polypeptide is substantially free fromother biological macromolecules. The substantially pure polypeptide isat least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight.Purity can be measured by any appropriate standard method, for example,by column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

[0060] The “percent identity” of two amino acid sequences or of twonucleic acids is determined using the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLASTN and BLASTX programs (version 2.0, 2.1and 2.2) of Altschul et al. (1990). J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the BLASTN program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the BLASTX program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Wheregaps exist between two sequences, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs for the determination ofpercent identity of amino acid sequences or nucleotide sequences, thedefault parameters of the respective programs can be used. The programsare available on the world wide web at: www.ncbi.nlm.nih.gov.

[0061] As used herein, the term “transgene” means a nucleic acidsequence (encoding, e.g., one or more subject polypeptides), which ispartly or entirely heterologous, i.e., foreign, to the transgenic plant,animal, or cell into which it is introduced, or, is homologous to anendogenous gene of the transgenic plant, animal, or cell into which itis introduced, but which is designed to be inserted, or is inserted,into the plant's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can include one or more transcriptionalregulatory sequences and other nucleic acid sequences, such as introns,that may be necessary for optimal expression of the selected nucleicacid, all operably linked to the selected nucleic acid, and may includean enhancer sequence.

[0062] As used herein, the term “transgenic cell” refers to a cellcontaining a transgene.

[0063] As used herein, a “transgenic plant” is any plant in which one ormore, or all, of the cells of the plant includes a transgene. Thetransgene can be introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by T-DNA mediated transfer, electroporation, orprotoplast transformation. The transgene may be integrated within achromosome, or it may be extrachromosomally replicating DNA.

[0064] As used herein, the term “tissue-specific promoter” means a DNAsequence that serves as a promoter, i.e., regulates expression of aselected DNA sequence operably linked to the promoter, and which effectsexpression of the selected DNA sequence in specific cells of a tissue,such as a leaf, root, seed, or stem.

[0065] As used herein, the terms “hybridizes under stringent conditions”and “hybridizes under high stringency conditions” refers to conditionsfor hybridization in 6×sodium chloride/sodium citrate (SSC) buffer atabout 45° C., followed by two washes in 0.2×SSC buffer, 0.1% SDS at 60°C. or 65° C. As used herein, the term “hybridizes under low stringencyconditions” refers to conditions for hybridization in 6×SSC buffer atabout 45° C., followed by two washes in 6×SSC buffer, 0.1% (w/v) SDS at50° C.

[0066] A “heterologous promoter”, when operably linked to a nucleic acidsequence, refers to a promoter which is not naturally associated withthe nucleic acid sequence.

[0067] As used herein, an agent with “anthelmintic activity” is anagent, which when tested, has measurable nematode-killing activity orresults in reduced fertility or sterility in the nematodes such thatfewer viable or no offspring result, or compromises the ability of thenematode to infect or reproduce in its host, or interferes with thegrowth or development of a nematode. The agent may also display nematoderepellant properties. In the assay, the agent is combined withnematodes, e.g., in a well of microtiter dish, in liquid or solid mediaor in the soil containing the agent. Staged adult nematodes are placedon the media. The time of survival, viability of offspring, and/or themovement of the nematodes are measured. An agent with “anthelminticactivity” can, for example, reduce the survival time of adult nematodesrelative to unexposed similarly staged adults, e.g., by about 20%, 40%,60%, 80%, or more. In the alternative, an agent with “anthelminticactivity” may also cause the nematodes to cease replicating,regenerating, and/or producing viable progeny, e.g., by about 20%, 40%,60%, 80%, or more. The effect may be apparent immediately or insuccessive generations.

[0068] As used herein, the term “binding” refers to the ability of afirst compound and a second compound that are not covalently linked tophysically interact. The apparent dissociation constant for a bindingevent can be 1 mM or less, for example, 10 nM, 1 nM, and 0.1 nM or less.

[0069] As used herein, the term “binds specifically” refers to theability of an antibody to discriminate between a target ligand and anon-target ligand such that the antibody binds to the target ligand andnot to the non-target ligand when simultaneously exposed to both thegiven ligand and non-target ligand, and when the target ligand and thenon-target ligand are both present in molar excess over the antibody.

[0070] As used herein, the term “altering an activity” refers to achange in level, either an increase or a decrease in the activity,(e.g., an increase or decrease in the ability of the polypeptide to bindor regulate other polypeptides or molecules) particularly a fatty aciddesaturase-like or fatty acid desaturase activity (e.g., the ability tointroduce a double bond at the delta-12 position of a fatty acid). Thechange can be detected in a qualitative or quantitative observation. Ifa quantitative observation is made, and if a comprehensive analysis isperformed over a plurality of observations, one skilled in the art canapply routine statistical analysis to identify modulations where a levelis changed and where the statistical parameter, the p value, is, forexample, less than 0.05.

[0071] Unless otherwise specified, a “substituted” carbon, carbon chain,or methyl, alkyl can have one or more hydrogens replaced by anothergroup, e.g., a halogen or a hydroxyl group.

[0072] In part, the nematicidal fatty acid analog described hereinprovide an effective, environmentally safe means of inhibiting nematodemetabolism, growth, viability, fecundity, development, infectivityand/or the nematode life-cycle. The compounds may be used alone or incombination with other nematicidal agents. The reduced phyto-toxicity atactive concentrations (i.e., greater therapeutic window) of many of thecompounds of the invention compared to prior art nematicidal fatty acidcompounds allows for application post-planting and reduced handlingcosts providing economic incentives in addition to the environmentalbenefits.

[0073] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

[0074]FIG. 1 is a photograph of C. elegans grown on oleic acid methylester.

[0075]FIG. 2 is a photograph of C. elegans grown on linoleic acid methylester.

[0076]FIG. 3 is a photograph of C. elegans grown on ricinoleic acidmethyl ester.

[0077]FIG. 4 is a photograph of C. elegans grown on vernolic acid methylester.

[0078]FIG. 5 is a set of drawings depicting the structures of ricinoleicacid methyl ester, ricinelaidic acid methyl ester,12-oxo-9(Z)-octadecenoic acid methyl ester, crepenynic acid methylester, and vernolic acid methyl ester. The numbering of the carbons isindicated with the carbonyl carbon being carbon 1.

DETAILED DESCRIPTION

[0079] Described below are experiments demonstrating that delta-12 fattyacid desaturase activity is essential for nematode viability. Alsodescribed below are certain nematicidal fatty acid analogs, includingnematicidal fatty acid analogs that have activity consistent with thatof an inhibitor of a delta-12 fatty acid desaturase.

[0080] Unsaturated fatty acids are essential to the proper functioningof biological membranes. At physiological temperatures, polarglycerolipids that contain only saturated fatty acids cannot form theliquid-crystalline bilayer that is the fundamental structure ofbiological membranes. The introduction of an appropriate number ofdouble bonds (a process referred to as desaturation) into the fattyacids of membrane glycerolipids decreases the temperature of thetransition from the gel to the liquid-crystalline phase and providesmembranes with necessary fluidity. Fluidity of the membrane is importantfor maintaining the barrier properties of the lipid bilayer and for theactivation and function of certain membrane bound enzymes. There is alsoevidence that unsaturation confers some protection to ethanol andoxidative stress, suggesting that the degree of unsaturation of membranefatty acids has importance beyond temperature adaptation. Unsaturatedfatty acids are also precursors of polyunsaturated acids (PUFAs)arachidonic and eicosapentaenoic acids in animals, which are importantsources of prostaglandins. These molecules are local hormones that alterthe activities of the cells in which they are synthesized and inadjoining cells, mediating processes in reproduction, immunity,neurophysiology, thermobiology, and ion and fluid transport.

[0081] The ability of cells to modulate the degree of unsaturation intheir membranes is primarily determined by the action of fatty aciddesaturases. Desaturase enzymes introduce unsaturated bonds at specificpositions in their fatty acyl chain substrates, using molecular oxygenand reducing equivalents from NADH (or NADPH) to catalyze the insertionof double bonds. In many systems, the reaction uses a short electrontransport chain consisting of NAD(P)H, cytochrome b5 reductase, andcytochrome b5, to shuttle electrons from NAD(P)H and the carbon-carbonsingle bond to oxygen, forming water and a double bond (C═C). Manyeukaryotic desaturases are endoplasmic reticulum (ER) bound non-hemediiron-oxo proteins which contain three conserved histidine-rich motifsand two long stretches of hydrophobic residues. These hydrophobic alphahelical domains are thought to position the protein with its bulkexposed to the cytosolic face of the ER and to organize the active sitehistidines to appropriately coordinate the active diiron-oxo moiety.

[0082] While most eukaryotic organisms, including mammals, can introducea double bond into an 18-carbon fatty acid at the Δ9 position, mammalsare incapable of inserting double bonds at the Δ12 or Δ15 positions. Forthis reason, linoleate (18:2 Δ9,12) and linolenate (18:3 Δ9,12,15) mustbe obtained from the diet and, thus, are termed essential fatty acids.These dietary fatty acids come predominately from plant sources, sinceflowering plants readily desaturate the Δ12 and the Δ15 positions.Certain animals, including some insects and nematodes, can synthesize denovo all their component fatty acids including linoleate and linolenate.The nematode C. elegans, for example, can synthesize de novo a broadrange of polyunsaturated fatty acids including arachidonic acid andeicosapentaenoic acids, a feature not shared by either mammals orflowering plants (Spychalla et al. (1997) Proc. Natl. Acad. Sci USA94(4):1142-7).

[0083] The C. elegans desaturase gene fat-2 has been expressed in S.cerevisiae and shown to be delta-12 fatty acid desaturase (Peyou-Ndi etal. (2000) Arch. Biochem. Biophys. 376(2):399-408). This enzymeintroduces a double bond between the 12th and the 13th carbons (from thecarboxylate end) and can convert the mono-unsaturated oleate (18:1 Δ9)and palmitoleate (16:1 Δ9) to the di-unsaturated linoleate (18:2 Δ9,12)and 16:2 Δ9,12 fatty acids, respectively.

[0084] The nematode delta-12 enzymes are potentially good targets foranti-nematode compounds for several reasons. Firstly, as mentionedabove, mammals are thought not to have delta-12 fatty acid desaturases.In addition, the enzymes appear to be phylogenetically diverged fromtheir homologs in plants, having less than 40% pairwise sequenceidentity at the amino acid level and phylogenetic analyses demonstrateclustering of nematode delta-12 and ω-3 desaturases away from homologsin plants. Experiments with both transgenic Arabidopsis and soybeansreveal that plants can tolerate significant reductions in linoleate orlinolenate, suggesting that inhibitors of delta-12 desaturases wouldlikely not be toxic to plants (Miquel & Browse (1992) J. Biol. Chem.267(3):1502-9; Singh et al. (2000) Biochem. Society Trans. 28: 940-942;Lee et al. (1998) Science 280:915-918). Thus, inhibitors of the enzymeare likely to be non-toxic to mammals. Importantly, as detailed herein,a delta-12 fatty acid desaturase of nematodes has been shown to beessential to their viability, both through inhibitor and RNA-mediatedinterference studies. Thus, delta-12 fatty acid desaturases could serveas ideal targets for anti-nematode control, as inhibitors of the enzymecould specifically target nematodes while leaving their animal and planthosts unharmed.

[0085] Numerous analogs of fatty acids exist and some may act asspecific inhibitors of enzymes such as desaturases that act on fattyacids, a fact that could be exploited for development of anti-nematodecompounds. Sterculic acid, a cyclopropenoid fatty acid analog of oleicacid, is a potent inhibitor of delta-9 fatty acid desaturases (Schmid &Patterson (1998) Lipids 23(3):248-52; Waltermann & Steinbuchel (2000)FEMS Microbiol Lett.190(1):45-50). It has also been speculated thatcyclopropenoid analogs of linoleic acid may similarly inhibit delta-12fatty acid clesaturases (Dulayynmi et al. (1997) Tetrahedron53(3):1099-1110). It is worth noting however that malvalate, a delta-8cyclopropene fatty acid, seems to be equally inhibitory to delta-9desaturases in some systems, as the delta-9 cyclopropene fatty acidsterculate (Schmid & Patterson (1998) Lipids 23(3):248-52),demonstrating how difficult it is to predict inhibitory profiles forsome fatty acid analogs. Thia fatty acid analogs are also potentialinhibitors of fatty acid desaturases (Skrede et al. (1997) BiochimBiophys Acta 1344(2):115-131; Hovik et al. (1997) Biochim Biophys Acta1349(3):251-256) as are trans fatty acids (Choi et al. (2001) BiochemBiophys Res Commun 284(3):689-93). However, the specificity andpesticidal activity of these analogs is again difficult to predict(Beach et al. (1989) Mol Biochem Parasitol 35(1):57-66).

[0086] Certain fatty acids are also specific receptor antagonists(Yagaloff(1995) Prostaglandins Leukot Essent Fatty Acids 52(5):293-7).

[0087] Other analogs of linoleic acid that may also be specific delta-12inhibitors include the epoxy fatty acid (vernolic acid), the acetylenicfatty acid (crepenynic acid), 12-oxo-9(Z)-octadecenoic acid methyl esteror the hydroxy fatty acids (ricinoleic and ricinelaidic acid).Inhibitors that interfere with delta-12 fatty acid desaturase activityare expected to be toxic to nematodes. Importantly, fatty acid analogssuch as ricinoleic, ricinelaidic, vernolic and crepenynic acid methylesters do not appear to be toxic (or are very much less toxic) to atleast some plants and are predicted not to be toxic (or are very muchless toxic) to at least some animals, including mammals. Such fatty acidanalogs could potentially be used in the development of nematode controlagents.

[0088] Although previously expressed in plants, fatty acid analogs suchas crepenynate, ricinoleate and vernolate acids were not thought to bespecific inhibitors of the endogenous delta-12 desaturase desaturase(Broun & Somerville (1997) Plant. Physiol. 113:933-942; Singh et al.(2000) Biochem. Society Trans. 28(6): 940-942). Changes in the ratio ofoleate to linoleate in plants expressing the genes for these analogs wasinstead attributed to a negative interaction between the enzymesinvolved (Singh et al. (2001) Planta 212: 872-879). Addition ofricinoleate exogenously to Neurospora crassa results in a significantdecrease in oleate (C_(1 8:1)) and an increase in linolenate (C18:3)again providing no indication that compounds like ricinoleate were infact specific delta-12 desaturase inhibitors (Goodrich-Tanrikulu et al.(1996) Appl Microbiol Biotechnol. 46(4):382-7).

[0089] We made the surprising discovery that methyl esters of certainfatty acid analogs (e.g., ricinoleate, vernolate) are nematicidal andhave activity consistent with that of specific inhibitors of nematodedelta-12 desaturases. The fatty acid methyl esters show significantlyenhanced activity over other eighteen carbon fatty acid esters such asoleate, elaidate and linoleate. In contrast to short chain seeminglynon-specific pesticidal fatty acid esters such as laurate andpelargonate, the fatty acid analogs that are predicted delta-12desaturase inhibitors show dramatically reduced phytoxicity and cantherefore be used effectively while minimizing undesirable damage tonon-target organisms.

[0090] Fatty acid analogs or other types of inhibitors may be suppliedto plants exogenously, through sprays for example. The fatty acidanalogs may also be applied as a seed coat. It is also possible toprovide inhibitors through a host organism or an organism on which thenematode feeds. The host organism or organism on which the nematodefeeds may or may not be engineered to produce lower amounts oflinoleate. For example, a host cell that does not naturally produce aninhibitor of a nematode fatty acid desaturase-like polypeptide can betransformed with genes encoding enzymes capable of making inhibitoryanalogs and provided with appropriate precursor chemicals exogenously ifnecessary. Alternatively, the active inhibitors and precursors can bemade endogenously by the expression of the appropriate enzymes. Inaddition, yeast or other organisms can be modified to produceinhibitors. Nematodes that feed on such organisms would then be exposedto the inhibitors.

[0091] In one embodiment, transgenic cells and/or organisms could begenerated that produce enzymes active on fatty acids (e.g.,desaturating, hydroxylating, and/or epoxygenating enzymes). Such enzymesmay be expressed, for example, in plants, vertebrates, and/or nematodes.These enzymes may produce fatty acids, analogs, or other inhibitors thatcan then act as specific inhibitors for other enzymes such as a fattyacid desaturase (e.g., a delta-12 epoxygenase from Crepis palaestinaproduces vernolic acid in transgenic Arabidopsis) (Singh et. al. (2000)Biochem. Society Trans. 28:940-942; Lee et al. (1998) Science280:915-918).

[0092] The fatty acid analogs used in the invention can be applied toanimals, plants or the environment of plants needing nematode control orto the food of animals needing nematode control. The compositions may beapplied by, for example drench or drip techniques. With dripapplications fatty acid analogs can be applied directly to the base ofthe plants or the soil immediately adjacent to the plants. Thecomposition may be applied through existing drip irrigation systems.This procedure is particularly applicable for cotton, strawberries,tomatoes, potatoes, vegetables and ornamental plants. Alternatively, adrench application can be used where a sufficient quantity ofnematicidal composition is applied such that it drains to the root areaof the plants. The drench technique can be used for a variety of cropsand turf grasses. The drench technique can also be used for animals.Preferably, the nematicidal compositions would be administered orally topromote activity against internal parasitic nematodes. Nematicidalcompositions may also be administered in some cases by injection of thehost animal.

[0093] In a preferred embodiment of the subject invention, a compound ofthe invention will be applied as an aqueous micro-emulsion. Theconcentration of the nematicidal composition should be sufficient tocontrol the nematode without causing phytotoxicity to the desired plantor undue toxicity to the animal host. An important aspect of theinvention is the surprising discovery that certain fatty acid analogs(e.g., ricinoleate, ricinelaidate, vernolate) that are predicted to bespecific inhibitors of nematode delta-12 desaturases are nematicidal andshow significantly enhanced activity over non-specific pesticidal fattyacid esters such as oleate, elaidate and linoleate. Moreover, thecompounds show reduced phytotoxicity compared to non-specific shortchain pesticidal fatty acid esters such as laurate and pelargonate.Thus, the compositions of this invention show excellent nematicidalactivity at concentrations that are not phytotoxic.

[0094] The nematicidal fatty acid analogs of the invention can beapplied in conjunction with another nematicidal agent. The second agentmay, for example, be applied simultaneously or sequentially. Suchnematicidal agents can include for example, avermectins for animalapplications.

[0095] A nematicidal fatty acid analog may also be coupled to an agentsuch as glyphosate to improve phloem mobility to the roots of plants.

[0096] The aforementioned nematicidal fatty acid ester compositions canbe used to treat diseases or infestations caused by nematodes of thefollowing non-limiting, exemplary genera: Anguina, Ditylenchus,Tylenchorhynchus, Pratylenchus, Radopholus, Hirschmanniella, Nacobbus,Hoplolaimus, Scutellonema, Rotylenchus, Helicotylenchus, Rotylenchulus,Belonolaimus, Heterodera, other cyst nematodes, Meloidogyne,Criconemoides, Hemicycliophora, Paratylenchus, Tylenchulus,Aphelenchoides, Bursaphelenchus, Rhadinaphelenchus, Longidorus,Xiphinema, Trichodorus, and Paratrichoclorus, Dirofiliaria, Onchocerca,Brugia, Acanthocheilonema, Aelurostrongylus, Anchlostoma,Angiostrongylus, Ascaris, Bunostomum, Capillaria, Chabertia, Cooperia,Crenosoma, Dictyocaulus, Dioctophyme, Dipetalonema, Dracunculus,Enterobius, Filaroides, Haemonchus, Lagochilascaris, Loa, Manseonella,Muellerius, Necator, Nematodirus, Oesophagostomum, Ostertagia,Parafilaria, Parascaris, Physaloptera, Protostrongylus, Setaria,Spirocerca, Stephanogilaria, Strongyloides, Strongylus, Thelazia,Toxascaris, Toxocara, Trichinella, Trichostrongylus, Trichuris,Uncinaria, and Wuchereria. Particularly preferred are nematodesincluding Dirofilaria, Onchocerca, Brugia, Acanthocheilonema,Dipetalonemna, Loa, Mansonella, Parafilaria, Setaria, Stephanofilaria,and Wucheria, Pratylenchus, Heterodera, Meloidogyne, Paratylenchus.Species that are particularly preferred are: Ancylostoma caninum,Haemonchus contortus, Trichinella spiralis, Trichurs muris, Dirofilariaimmitis, Dirofilaria tenuis, Dirofilaria repens, Dirofilari ursi,Ascaris suum, Toxocara canis, Toxocara cati, Strongyloides ratti,Parastrongyloicles trichosuri, Heterodera glycines, Globodera pallida,Meloidogyne javanica, Meloiclogyne incognita, and Meloiclogyne arenaria,Radopholus similis, Longicdorus elongatus, Meloidogyne hapla, andPratylenchus penetrans.

[0097] The following examples are, therefore, to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. All of the publications cited herein are herebyincorporated by reference in their entirety.

EXAMPLE 1 RNA Mediated Interference (RNAi)

[0098] A double stranded RNA (dsRNA) molecule can be used to inactivatea delta-12 fatty acid desaturase (delta-12 fat-2) gene in a cell by aprocess known as RNA mediated-interference (Fire et al. (1998) Nature391:806-811, and Gönczy et al. (2000) Nature 408:331-336). The dsRNAmolecule can have the nucleotide sequence of a delta-12 fat-2 nucleicacid (preferably exonic) or a fragment thereof. The dsRNA molecule canbe delivered to nematodes via direct injection, or by soaking nematodesin aqueous solution containing concentrated dsRNA, or by raisingbacteriovorous nematodes on E. coli genetically engineered to producethe dsRNA molecule.

[0099] RNAi by injection: To examine the effect of inhibiting delta-12fat-2 activity, a dsRNA corresponding to the C. elegans delta-12 fat-2gene was injected into the nematode, basically as described in Mello etal. (1991) EMBO J. 10:3959-3970. Briefly, a plasmid was constructed thatcontains a portion of the C. elegans delta-12 fat-2 sequence,specifically a fragment 651 nucleotides long, containing the entirefirst exon and terminating just before the conserved intron splicejunction between the first exon and first intron. This construct encodesapproximately the first 217 amino acids of the C. elegans delta-12 fat-2gene. Primers were used to specifically amplify this sequence as alinear dsDNA. Single-stranded RNAs were transcribed from these fragmentsusing T7 RNA polymerase and SP6 RNA polymerase (the RNAs correspond tothe sense and antisense RNA strands). RNA was precipitated andresuspended in RNAse free water. For annealing of ssRNAs to form dsRNAs,ssRNAs were combined, heated to 95° for two minutes then allowed to coolfrom 70° to room temperature over 1.5-2.5 hours.

[0100] DsRNA was injected into the body cavity of 15-20 young adult C.elegans hermaphrodites. Worms were immobilized on an agarose pad andtypically injected at a concentration of 1 mg/ml. Injections wereperformed with visual observation using a Zeiss Axiovert compoundmicroscope equipped with 10× and 40×DIC objectives, for example. Needlesfor microinjection were prepared using a Narishige needle puller, stagemicromanipulator (Leitz) and a N2-powered injector (Narishige) set at10-20 p.s.i. After injection, 200 μl of recovery buffer (0.1% salmonsperm DNA, 4% glucose, 2.4 mM KCl, 66 mM NaCl, 3 mM CaCl2, 3 mM HEPES,pH 7.2) were added to the agarose pad and the worms were allowed torecover on the agarose pad for 0.5-4 hours. After recovery, the wormswere transferred to NGM agar plates seeded with a lawn of E. coli strainOP50 as a food source. The following day and for 3 successive daysthereafter, 7 individual healthy injected worms were transferred to newNGM plates seeded with OP50. The number of eggs laid per worm per dayand the number of those eggs that hatch and reach fertile adulthood weredetermined. As a control, Green Fluorescent Protein (GFP) dsRNA wasproduced and injected using similar methods. GFP is a commonly usedreporter gene originally isolated from jellyfish and is widely used inboth prokaryotic and eukaryotic systems. The GFP gene is not present inthe wild-type C. elegans genome and, therefore, GFP dsRNA does nottrigger an RNAi phenotype in wild-type C. elegans. The C. elegansdelta-12 FAT RNAi injection phenotype presented as a strongly reduced F1hatch-rate, with the few surviving individuals arrested in an earlylarval stage.

[0101] RNAi by feeding: C. elegans can be grown on lawns of E. coligenetically engineered to produce double stranded RNA (dsRNA) designedto inhibit delta-12 fat-2 expression. Briefly, E. coli were transformedwith a genomic fragment of a portion of the C. elegans fat-2 genesequence, specifically a fragment 651 nucleotides long, containing theentire first exon and terminating just before the conserved intronsplice junction between the first exon and first intron. This constructencodes approximately the first 217 amino acids of the C. elegansdelta-12 FAT gene. The 651 nucleotide genomic fragment was cloned intoan E. coli expression vector between opposing T7 polymerase promoters.The clone was then transformed into a strain of E. coli that carries anIPTG-inducible T7 polymerase. As a control, E. coli was transformed witha gene encoding the Green Fluorescent Protein (GFP). Feeding RNAi wasinitiated from C. elegans eggs or from C. elegans L4s. When feeding RNAiwas started from C. elegans eggs at 23° C. on NGM plates containing IPTGand E. coli expressing the C. elegans delta-12 FAT or GFP dsRNA, the C.elegans delta-12 FAT RNAi feeding phenotype presented as partiallysterile F1 individuals and dead F2 embryos. When feeding RNAi wasstarted from C. elegans L4 larvae at 23° C. on NGM plates containingIPTG and E. coli expressing the C. elegans DELTA-12 FAT or GFP dsRNA,the C. elegans RNAi feeding phenotype presented as partially sterile P0individuals (i.e., the individuals exposed initially) withdevelopmentally arrested, sterile F1 nematodes. The sequence of thefat-2 gene is of sufficiently high complexity (i.e., unique) such thatthe RNAi is not likely to represent cross reactivity with other genes.

[0102]C. elegans cultures grown in the presence of E. coli expressingdsRNA and those injected with dsRNA from the delta-12 FAT gene werestrongly impaired indicating that the fatty acid desaturase-like geneprovides an essential function in nematodes and that dsRNA from thefatty acid desaturase-like gene is lethal when ingested by or injectedinto C. elegans.

EXAMPLE 2 Rescue of C. elegans DELTA-12 FAT RNAi Feeding Phenotype byLinoleic Acid Methyl Ester

[0103] The C. elegans delta-12 fatty acid desaturase (FAT-2 protein)converts the mono-unsaturated oleic acid to the di-unsaturated fattyacid linoleic acid. The delta-12 FAT RNAi prevents expression of thedelta-12 fatty acid desaturase, which is predicted to cause a decreasein levels of linoleic acid in the nematode, leading to arresteddevelopment and death. Addition of 3 mM linoleic acid methyl ester tothe NGM media used for the RNAi experiment brings about a partial rescueof the delta-12 FAT RNAi feeding phenotype. Addition of 3 mM oleic acidmethyl ester does not rescue the delta-12 FAT RNAi feeding phenotype(see Table I below). TABLE 1 C. elegans delta-12 fat-2 RNAi feedingphenotypes (starting with C. elegans L4 larvae as the P0 animal) FattyAcid Added P0 phenotype F1 phenotype F2 phenotype None SeverelyDevelopmentally NA reduced egg arrested and laying (almost sterilesterile) Oleic Acid Severely Developmentally NA Methyl Ester reduced eggarrested and laying (almost sterile sterile) Linoleic Acid Reduced eggModerately delayed Slightly Methyl Ester laying development and delayedmoderately reduced development egg laying

EXAMPLE 3 Preparation of Caenorhabditis elegans and Fatty Acid MethylEsters

[0104] Mixed stage Caenorhabditis elegans were washed off plates seededwith OP50 bacteria using M9 solution. 250 μl of the M9 solution, whichcontained about 50 worms, was pipetted into each well of a 24-wellplate.

[0105] Fatty acid methyl ester emulsions were prepared following theteachings of Kim et al (U.S. Pat. No. 5,698,592). Briefly, 1 ml 1% stocksolution emulsions were prepared by mixing 10 μl of fatty acid methylester with 20 μl of the surfactant Igepal CO 630 in a 1.5 ml eppendorftube. After careful mixing of fatty acid and Igepal CO 630, 850 μl ofddH20 was added and mixed by gentle pipetting until a homogeneoussolution was obtained. Finally, 120 μl of pure isopropanol was added andmixed by gentle pipetting. This stock solution was then used to producevarious fatty acid methyl ester dilution emulsions in 24-well plateassays.

EXAMPLE 4 Nematicidal Activity of Single Fatty Acid Methyl EsterEmulsions Against Caenorhabditis elegans

[0106] To each well, fatty acid emulsions or control emulsions wereadded and rapidly mixed by swirling. Nematode viability was scored byvisual observation and motility assays at various time points 24 hoursfollowing addition of emulsions or controls. The fatty acid emulsionstested were methyl esters of nonanoic (pelargonic) acid, ricinoleicacid, vernolic acid, linoleic acid, oleic acid, and control emulsionslacking fatty acids.

[0107] The structures of ricinoleic acid methyl ester, crepenynic acidmethyl ester and vernolic acid methyl ester are depicted in FIG. 5.TABLE 2 Nematicidal activity of fatty acid methyl ester emulsionsagainst C. elegans Percentage of Worm Death Fatty Acid Concentration 1hr 6 hr 24 hr Nonanoic 0.1% 100% 100% 100% (C9-methyl ester) 0.003%  50%  50%  50% Ricinoleic Acid 0.1%  90%  90%  90% (C18-methyl ester)0.003%   60%  60%  60% Vernolic Acid 0.1%  65%  65%  75% (C18-methylester) 0.003%   20%  20%  20% Linoleic Acid 0.1% 0-5% 0-5% 0-5%(C18-methyl ester) 0.003%  0-5% 0-5% 0-5% Oleic Acid 0.1% 0-5% 0-5% 0-5%(C18-methyl ester) 0.003%  0-5% 0-5% 0-5% Control 0.1% 0-5% 0-5% 0-5%(no methyl ester) 0.003%  0-5% 0-5% 0-5%

[0108] Both nonanoic and ricinoleic acid methyl ester emulsions arestrongly nematicidal at a concentration of 0.1%. Nonanoic methyl esteremulsions cause an almost immediate cessation of nematode movement andsubsequent death whereas ricinoleic methyl ester emulsions require up to30 minutes before strong killing effects are apparent. However, at0.003%, nonanoic acid methyl ester emulsions temporarily “stunned” C.elegans, initially giving the appearance of a 100% death phenotype.Several hours post inoculation, many nematodes recover and start movingagain. This “stun” effect was not observed with the other fatty acidemulsions.

EXAMPLE 5 Preparation of Root Knot Nematode J2 Larvae (Meloidogyne spp.)

[0109] Meloidogyne incognita and javanica were prepared from tomatoroots. The roots were bleached and the debris was separated from the J2larvae and eggs by filtration followed by sucrose density gradientcentrifugation. Eggs were hatched over 4 days at 15° C. and the J2larvae were collected by passage though a filter, followed bycentrifugation.

EXAMPLE 6 Nematicidal Activity of Fatty Acid Methyl Ester EmulsionsAgainst Root Knot Nematodes (Meloidogyne spp.)

[0110] Nematodes and emulsions were incubated with shaking at roomtemperature for 48 hours. The contents of each well were transferred toa small spot on individual NGM plates lacking bacteria. About 24 hoursafter the transfer to plates, worms on and off the inoculation spot werecounted as not viable or viable, respectively. Worms were consideredviable if they had crawled away from the inoculation spot, or if theywere moving. Worms were considered non-viable if they remained at theinoculation spot. TABLE 3 Nematicidal activity of fatty acid methylester emulsions against M. javanica and M. incognita Fatty acid M.javanica M. incognita (0.1%) (% not viable) (% not viable) Vernolic Acid90% 100% (C18-methyl ester) Nonanoic 100%  100% (C9-methyl ester)Ricinoleic Acid 60%  95% (C18-methyl ester) Oleic Acid 20%  25%(C18-methyl ester)

[0111] Nonanoic, vernolic and ricinoleic acid methyl ester emulsionshave significant nematicidal activity against root knot nematodes(Meloidogyne spp.) at a concentration of 0.1%.

EXAMPLE 7 Phytotoxicity Evaluations of Fatty Acid Methyl Esters

[0112] Sterilized tomato seeds were germinated in magenta jarscontaining Gamborg's agar media. After two weeks of growth, seedlingswere treated with 250 μl of 1% fatty acid methyl ester emulsion(nonanoic acid, ricinoleic acid, ricinelaidic acid, oleic acid, or acontrol emulsion lacking any fatty acid), applied directly to thestem-media interface. Tomato seedlings were scored at various timesafter application of emulsions. Of the fatty acids tested, only 1%nonanoic acid methyl ester emulsion showed obvious effects on thetomatoes. Within 18 hours of nonanoic acid emulsion application, thosetomatoes showed a distinct loss of turgor pressure (wilting phenotype)and had become noticeably less green in appearance. Within 24 hours,nonanoic acid treated tomatoes were almost entirely bleached to a palewhite color and had nearly totally collapsed with most leaves lyingdirectly on the agar media surface. Importantly, none of the tomatoestreated with the other fatty acid methyl ester emulsions showed visibleeffects. Therefore, ricinoleic and ricinelaidic (see examples 8-11) acidmethyl esters show excellent potential as anthelmintic chemicals basedon their combination of high nematicidal properties and with favorablelow phytotoxicity.

EXAMPLE 8 Nematicidal Activity of Single Fatty Acid Methyl EsterEmulsions Against a Spectrum of Free-Living, Animal Parasitic, and PlantParasitic Nematodes

[0113] Briefly, the indicated fatty acid emulsions were added andrapidly mixed by swirling. Nematode viability was scored by visualobservation and motility assays 24 hours following addition of emulsions(48 hours for plant parasitic nematodes Meloidogyne and Heteroderaspecies). The fatty acid emulsions tested were methyl esters of nonanoic(pelargonic) acid, ricinelaidic acid, ricinoleic acid, vernolic acid,linoleic acid, and oleic acid. Results for fatty acid emulsions againstfree living, animal parasitic, and plant parasitic nematodes arecombined in one table to facilitate comparison of different emulsionactivities against nematodes exhibiting diverse lifestyles. Resultsshown are mean % values obtained from multiple independent experimentsTABLE 4 Nematicidal activity of various fatty acid methyl esters againstvarious free-living, animal parasitic, and plant parasitic nematodes %Worm Death (24 hr) − control Inhibitors + control Worm (% solution)Oleic Linoleic Vernolic Ricinoleic Ricinelaidic Nonanoic C. elegans(0.1%) <10 <10 80 100 100 100 C. elegans (0.01%) <10 <10 50 80 100 100C. elegans (0.001%) <10 30 30 75 30 P. trichosuri (0.1%) ˜10 ˜25 ˜95 ˜50100 P. trichosuri (0.01%) ˜10 ˜25 ˜90 ˜60 100 P. trichosuri (0.001%) M.incognita (0.1%) 20 98 95 ˜99 100 M. incognita (0.01%) 20 73 83 ˜99 M.incognita (0.001%) 97 M. javanica (0.1%) 20 90 60 100 100 M. javanica(0.01%) 0-5 60 5 100 M. javanica (0.001%) ˜60 H. glycines (0.1%) <10 <2030 ˜60 100 100 H. glycines (0.01%) <10 <20 20 ˜60 100 >95 H. glycines(0.001%) <10 <20 18 ˜40 100 P. scribneri (0.1%) <20 <20 <20 <20 ˜70 <20P. scribneri (0.01%) <20 <20 <20 <20 ˜40 <20 P. scribneri (0.001%)

[0114] The Caenorhabiditis elegans were mixed stage populations. Similareffects seen on several other free-living nematode species. TheParastrongyloides trichosuri (parasite of Australian bushtail possum)were dauer-like infective 3^(rd) stage larva. Similar effects are alsoseen against free-living stages. The Meloidogyne incognita andMeloidogyne javanica (root knot nematode) were 2^(nd) stage juveniles(dauer-like infective stage). The Heterodera glycines (soybean cystnematode) were 2^(nd) stage juveniles (dauer-like infective stage).Finally, the Pratylenchus scribneri (corn lesion nematode) were mixedstage populations.

[0115] As the data in the table above demonstrate, both ricinelaidic andricinoleic acid methyl ester emulsions are strongly nematicidal atconcentrations of 0.1% and 0.01%. Ricinelaidic acid methyl ester inparticular showed favorable nematicidal activity against a wide spectrumof divergent nematode genera.

EXAMPLE 9 Nematicidal Activity of Single Fatty Acid Methyl EsterEmulsions made with Tween-20 (Replacing Igepal CO 630) AgainstCaenorhabditis elegans

[0116] Some nematodes assayed as described in Example 9 lacked toleranceto emulsions made with the surfactant Igepal CO 630. For this reasonsome assays were repeated with Tween-20-based emulsions.

[0117] Briefly, 1 ml 1% stock solution emulsions were prepared by mixing10 μl of fatty acid methyl ester with 20 μl of the surfactant Tween-20in a 1.5 ml eppendorf tube. After careful mixing of fatty acid andTween-20, 850 μl of ddH20 was added and mixed by gentle pipetting untila homogeneous solution was obtained. Finally, 120 μl of pure isopropanolwas added and mixed by gentle pipetting. This stock solution was thenused to produce various fatty acid methyl ester dilution emulsions in24-well plate assays. TABLE 5 Nematicidal activity of various fatty acidmethyl esters emulsions with Tween-20 against C. elegans % Worm Death(24 hr) − control Inhibitors + control Worm (% solution) Oleic LinoleicVernolic Ricinoleic Ricinelaidic Pelargonic C. elegans (0.1%) <10 100100 100 C. elegans (0.01%) <10 100 100 100 C. elegans (0.001%) 40 60 30

[0118] As shown in the Table above, fatty acid methyl ester emulsionsmade with tween-20 replacing igepal CO 630 exhibited comparablenematicidal activity to igepal-based emulsions.

EXAMPLE 10 Nematicidal Activity of Single Fatty Acid Methyl EsterEmulsions Made With Cyclodextrins (Replacing Isopropanol) AgainstCaenorhabditis elegans

[0119] In an effort to increase the bioavailability of fatty acid methylesters in emulsions, isopropanol was replaced with one of twocyclodextrins (Methyl-β-Cyclodextrin or 2-Hydroxypropyl-β-Cyclodextrin).TABLE 6 Nematicidal activity of various fatty acid methyl esteremulsions with cyclodextrins against C. elegans % Worm Death (24 hr) −control Inhibitors + control Worm (% solution) Oleic Linoleic VernolicRicinoleic Ricinelaidic Pelargonic C. elegans (0.1%) <10 >95 C. elegans(0.01%) <10 >80 C. elegans (0.001%) ˜50

[0120] Results for the most effective cyclodextrin emulsion formulation(10 μl fatty acid, 20 μl igepal CO 630, 60 μl2-Hydroxypropyl-β-Cyclodextrin, and 901 μl of H₂O) are shown above.

EXAMPLE 11 Nematicidal Activity of Fatty Acid Methyl Ester EmulsionsAgainst Animal Parasitic Microfilarial Nematodes (Brugia malayi)

[0121] To each well, tween-based fatty acid emulsions were added andrapidly mixed by swirling. Nematode microfilaria viability was scored byvisual observation of motility at 24 and 48 hours following addition ofemulsions. The fatty acid emulsions tested were methyl esters ofricinoleic acid, vernolic acid, ricinelaidic acid, oleic acid, andcontrol emulsions lacking fatty acids. The results of this study arepresented in Table 7 as the percentage of non-motile microfilaria 24hours after inoculation and in Table 8 as the percentage of non-motilemicrofilaria 48 hours after inoculation TABLE 7 Nematicidal activity offatty acid methyl ester emulsions against B. malayi 24 hrspost-inoculation Inhibitor 0.0% 0.0008% 0.0016% 0.003% 0.005% 0.0067%0.008% Ricinoleic 0 0 24.7 100 100 100 100 Vernolic 0 0 0 0 66 100 100Ricinelaidic 0 0 19.1 100 100 100 100 Oleic 0 0 0 0 0 0 0 Blank 0 0 0 00 0 0

[0122] TABLE 8 Nematicidal activity of fatty acid methyl ester emulsionsagainst B. malayi 48 hrs post-inoculation Inhibitor 0.0% 0.0008% 0.0016%0.003% 0.005% 0.0067% 0.008% Ricinoleic 0 0 64.2 100 100 100 100Vernolic 0 0 0 5 100 100 100 Ricinelaidic 0 0 49.4 100 100 100 100 Oleic0 0 0 0 0 0 0 Blank 0 0 0 0 0 0 0

[0123] As shown by the results in the Tables above, both ricinoleic andricinelaidic methyl esters exhibited strong microfilarialcidal activityagainst B. malayi (somewhat stronger than the activity of vernolic acidmethyl esters) in tween-based emulsions.

What is claimed is:
 1. A nematicidal composition comprising: (a) aneffective amount of a compound having the formula

wherein: R₁=a C₁-C₅ substituted or unsubstituted carbon chain, whereinthe substituants are selected from the group consisting of: hydroxy,oxo, halogen, amino, cyano, a singly or multiply substituted orunsubstituted C₁-C₂ carbon chain, cyclopropane, and epoxy; and R₂=aC₁₅-C₁₉ substituted or unsubstituted carbon chain having a cis or transdouble bond between the 9^(th) and 10^(th) carbons and either: (i) atriple bond between the 12^(th) and 13^(th) carbons or (ii) either asingle or double bond between the 12^(th) and 13^(th) carbons and atleast one substituant at one or both of the 12^(th) and 13^(th) carbons,wherein the substituants are selected from the group consisting ofhydroxy, oxo, halogen, amino, cyano, azido, a singly or multiplysubstituted or unsubstituted C₁-C₂ carbon chain, cyclopropane,cyclopropene, and epoxy; and (b) an aqueous surfactant.
 2. Thenematicidal composition of claim 1 wherein R₁=a C₁-C₅ substituted orunsubstituted carbon chain, wherein the substituants are selected fromthe group consisting of: hydroxy, oxo, halogen, amino, cyano, azido, anunsubstituted C₁-C₂ carbon chain, cyclopropane, and epoxy.
 3. Thenematicidal composition of claim 1 wherein R₂=a C₁₅-C₁₉ substituted orunsubstituted carbon chain having a cis or trans double bond between the9^(th) and 10^(th) carbons and either: (i) a triple bond between the12^(th) and 13^(th) carbons or (ii) either a single or double bondbetween the 12^(th) and 13^(th) carbons and at least one substituant atone or both of the 12^(th) and 13^(th) carbons, wherein the substituantsare selected from the group consisting of hydroxy, oxo, halogen, amino,cyano, azido, a unsubstituted C₁-C₂ carbon chain, cyclopropane,cyclopropene, and epoxy.
 4. The nematicidal composition of claim 1wherein the C₁-C₂ carbon chain of one or both of R₁ and R₂ issubstituted and the substituants are selected from the group consistingof: hydroxy, oxo, halogen, amino, cyano, azido, and epoxy.
 5. Thenematicidal composition of claim 1 wherein the C₁-C₂ carbon chain of oneor both of R₁ and R₂ is substituted and the substituants are selectedfrom the group consisting of: hydroxy, oxo, halogen, azido, and amino.6. The nematicidal composition of claim 1 wherein the C₁-C₂ carbon chainof R₁ is singly substituted.
 7. The nematicidal composition of claim 1wherein the C1-C2 carbon chain of R₂ is singly substituted.
 8. Thenematicidal composition of claim 1 wherein R₁=a substituted C₁ methyl.9. The nematicidal composition of claim 1 wherein R₁ is a C₁-C₂substituted or unsubstituted carbon chain.
 10. The nematicidalcomposition of claim 1 wherein R₂ is substituted only at one or both of12^(th) and 13^(th) carbons.
 11. The nematicidal composition of claim 10wherein R₂ is substituted only at the 12^(th) carbon
 12. The nematicidalcomposition of claim 10 wherein R₂ is substituted only at the 13^(th)carbon.
 13. The nematicidal composition of claim 1 wherein within R₂there is a triple bond between the 12^(th) and 13^(th) carbons.
 14. Thenematicidal composition of claim 10 wherein within R₂ the substituantsare selected from the group consisting of: hydroxy, oxo, epoxy, and a C₁methyl.
 15. A nematicidal composition comprising (a) a fatty acid methylester selected from the group consisting of: ricinoleic acid methylester, ricinelaidic acid methyl ester, 12-oxo-9(Z)-octadecenoic acidmethyl ester, crepenynic acid methyl ester, and vernolic acid methylester; and (b) an aqueous surfactant.
 16. The nematicidal composition ofclaim 1 or claim 15 wherein the aqueous surfactant is selected from thegroup consisting of: ethyl lactate, Tween 20 and Igepal CO
 630. 17. Thenematicidal composition of claim 1 or claim 15 wherein the compositionfurther comprises: (c) a permeation enhancer.
 18. The nematicidalcomposition of claim 17 wherein the permeation enhancer is acyclodextrin.
 19. The nematicidal composition of claim 1 where thecomposition further comprises: (c) a co-solvent.
 20. The nematicidalcomposition of claim 19 wherein the co-solvent is isopropanol.
 21. Thenematicidal composition of claim 1 or claim 15 further comprising anematicide selected from the group consisting of: avermectins,ivermectin, and milbemycin.
 22. A method for control of unwantednematodes, the method comprising administering to mammals, plants, seedsor soil a nematicidal composition comprising: (a) an effective amount ofa compound having the formula

wherein: R₁=a C₁-C₅ substituted or unsubstituted carbon chain, whereinthe substituants are selected from the group consisting of: hydroxy,oxo, halogen, amino, cyano, a substituted or unsubstituted C₁-C₂ carbonchain, cyclopropane, and epoxy; and R₂=a C₁₅-C₁₉ substituted orunsubstituted carbon chain having a cis or trans double bond between the9^(th) and 10^(th) carbons and either: (i) a triple bond between the12^(th) and 13^(th) carbons or (ii) either a single or double bondbetween the 12^(th) and 13^(th) carbons and at least one substituant atone or both of the 12^(th) and 13^(th) carbons, wherein the substituantsare selected from the group consisting of hydroxy, oxo, halogen, amino,cyano, azido, a singly or multiply substituted or unsubstituted C₁-C₂carbon chain, cyclopropane, cyclopropene, and epoxy; and (b) an aqueoussurfactant.
 23. The method of claim 22 wherein R₁=a C₁-C₅ substituted orunsubstituted carbon chain, wherein the substituants are selected fromthe group consisting of: hydroxy, oxo, halogen, amino, cyano, azido, aunsubstituted C₁-C₂ carbon chain, cyclopropane, and epoxy.
 24. Themethod of claim 22 wherein R₂=a C₁₅-C₁₉ substituted or unsubstitutedcarbon chain having a cis or trans double bond between the 9^(th) and10^(th) carbons and either: (i) a triple bond between the 12^(th and)13^(th) carbons or (ii) either a single or double bond between the12^(th) and 13^(th) carbons and at least one substituant at one or bothof the 12^(th) and 13^(th) carbons, wherein the substituants areselected from the group consisting of hydroxy, oxo, halogen, amino,cyano, a unsubstituted C₁-C₂ carbon chain, cyclopropane, cyclopropene,and epoxy.
 25. The method of claim 22 wherein the C₁-C₂ carbon chain ofone or both of R₁ and R₂ is substituted and the substituants areselected from the group consisting of: hydroxy, oxo, halogen, amino,cyano, azido, and epoxy.
 26. The method of claim 22 wherein the C₁-C₂carbon chain of one or both of R₁ and R₂ is substituted and thesubstituants are selected from the group consisting of: hydroxy, oxo,halogen, and amino.
 27. The method of claim 22 wherein the C₁-C₂ carbonchain of R₁ is singly substituted.
 28. The method of claim 14 whereinthe C₁-C₂ carbon chain of R₂ is singly substituted.
 29. The method ofclaim 14 wherein R₁ is a C₁-C₂ substituted or unsubstituted carbonchain.
 30. The method of claim 14 wherein R₂ is substituted only at oneor both of 12^(th) and 13^(th) carbons.
 31. The method of claim 14wherein R₂ is substituted only at the 12^(th) carbon.
 32. The method ofclaim 14 wherein R₂ is substituted only at the 13^(th) carbon.
 33. Themethod of claim 14 wherein within R₂ there is a triple bond between the12^(th) and 13^(th) carbons.
 34. The method of claim 14 wherein withinR₂ the substituants are selected from the group consisting of: hydroxy,oxo, epoxy, and a C₁ alkyl.
 35. A method for control of unwantednematodes, the method comprising administering to mammals, plants, seedsor soil a nematicidal composition comprising an effective amount of: (a)a fatty acid methyl ester selected from the group consisting of:ricinoleic acid methyl ester, ricinelaidic acid methyl ester,12-oxo-9(Z)-octadecenoic acid methyl ester, crepenynic acid methylester, and vernolic acid methyl ester; and (b) an aqueous surfactant.36. The method of claim 22 or claim 35 wherein the aqueous surfactant isselected from the group consisting of: ethyl lactate, Tween 20 andIgepal CO
 630. 37. The method of claim 22 or claim 35 wherein thecomposition further comprises: (c) a permeation enhancer.
 38. The methodof claim 37 wherein the permeation enhancer is a cyclodextrin.
 39. Themethod of claim 22 or 35 wherein the composition comprises: (c) aco-solvent.
 40. The method of claim 39 wherein the co-solvent isisopropanol.
 41. The method of claim 22 or claim 35 further comprisingadministering a nematicide selected from the group consisting of:avermectins, ivermectin, and milbemycin.
 42. The method of claim 22wherein the nematode infects plants and the nematicidal composition isapplied to the soil or to plants.
 43. The method of claim 42 wherein thenematicidal composition is applied to soil before planting.
 44. Themethod according to claim 42 where the nematicidal composition isapplied to soil after planting.
 45. The method of claim 42 wherein thenematicidal composition is applied to soil using a drip system.
 46. Themethod of claim 42 wherein the nematicidal composition is applied tosoil using a drench system.
 47. The method of claim 42 wherein thenematicidal composition is applied to plant roots.
 48. The method ofclaim 22 wherein the nematicidal composition is applied to seeds. 49.The method of claim 22 wherein the nematode infects a mammal.
 50. Themethod of claim 22 wherein the nematicidal composition is administeredto non-human mammal.
 51. The method of claim 22 wherein the nematicidalcomposition is administered to a human.
 52. The method of claim 50wherein the nematicidal composition is formulated as a drench to beadministered to a non-human animal.
 53. The method of claim 49 whereinthe nematicidal composition is formulated as an orally administereddrug.
 54. The method of claim 49 wherein the nematicidal composition isformulated as an injectable drug.
 55. A nematicidal feed for a non-humanmammal comprising: (a) a feed; (b) an effective amount of a nematicidalcompound having the formula

wherein: R₁=a C₁-C₅ substituted or unsubstituted carbon chain, whereinthe substituants are selected from the group consisting of: hydroxy,oxo, halogen, amino, cyano, a singly or multiply substituted orunsubstituted C₁-C₂ carbon chain, cyclopropane, and epoxy; and R₂=aC₁₅-C₁₉ substituted or unsubstituted carbon chain having a cis or transdouble bond between the 9^(th) and 10^(th) carbons and either: (i) atriple bond between the 12^(th) and 13^(th) carbons or (ii) either asingle or double bond between the 12^(th) and 13^(th) carbons and atleast one substituant at one or both of the 12^(th) and 13^(th) carbons,wherein the substituants are selected from the group consisting ofhydroxy, oxo, halogen, amino, cyano, azido, a singly or multiply orunsubstituted C₁-C₂ carbon chain, cyclopropane, cyclopropene, and epoxy;and (c) an aqueous surfactant.
 56. The nematicidal feed of claim 55wherein the feed has been treated to reduce linoleic acid content,linolenic acid content or both.
 57. The nematicidal feed of claim 56wherein both the gamma linolenic acid content and the alpha linolenicacid content have been reduced.
 58. The nematicidal feed of claim 55wherein the feed is selected from the group consisting of: soy, wheat,corn, sorghum, millet, alfalfa, clover, and rye.
 59. A nematicidalcomposition comprising: (a) an effective amount of a compound having theformula

wherein: R₁=a C₁-C₅ substituted or unsubstituted carbon chain, whereinthe substituants are selected from the group consisting of: hydroxy,oxo, halogen, amino, cyano, a singly or multiply substituted orunsubstituted C₁-C₂ carbon chain, cyclopropane, and epoxy; R₃=a C₁₁substituted or unsubstituted carbon chain having a cis or trans doublebond between the 9^(th) and 10^(th) carbons counting form the carbonylcarbon, wherein the substituants are selected from the group consistingof hydroxy, oxo, halogen, amino, cyano, a singly or multiply orunsubstituted C₁-C₂ carbon chain, cyclopropane, cyclopropene, and epoxy;R₄=a C₂-C₆ substituted or unsubstituted carbon chain wherein thesubstituants are selected from the group consisting of: hydroxy, oxo,halogen, amino, cyano, a singly or multiply substituted or unsubstitutedC₁-C₂ carbon chain, cyclopropane, and epoxy; X and Y are independently asubstituted or unsubstitued methyl or S, provided at least one or X andY is S and wherein the substituants on the methyl selected from thegroup consisting of: halogen, hydrogen, amino, oxo and hydroxy; and (b)an aqueous surfactant.
 60. The nematicidal composition of claim 59wherein one of X and Y is CH₂.
 61. A nematicidal composition comprising;(a) an effective amount of a compound having the formula

wherein: R₁=a C₁-C₅ substituted or unsubstituted carbon chain, whereinthe substituants are selected from the group consisting of: hydroxy,oxo, halogen, amino, cyano, a singly or multiply substituted orunsubstituted C₁-C₂ carbon chain, cyclopropane, and epoxy; and R₂=aC₁₅-C₁₉ substituted or unsubstituted carbon chain having a single bondbetween the 9^(th) and 10^(th) carbons and either: (i) a triple bondbetween the 12^(th) and 13^(th) carbons or (ii) either a single ordouble bond between the 12^(th) and 13^(th) carbons and at least onesubstituant at one or both of the 12^(th) and 13^(th) carbons, whereinthe substituants are selected from the group consisting of hydroxy, oxo,halogen, amino, cyano, azido, a substituted or unsubstituted C₁-C₂carbon chain, cyclopropane, cyclopropene, and epoxy; and (b) an aqueoussurfactant.
 62. The nematicidal composition of claim 61 wherein R₂=aC₁₅-C₁₉ substituted or unsubstituted carbon chain having a single bondbetween the 9^(th) and 10^(th) carbons and a single bond between the12^(th) and 13^(th) carbons and at least one substituant at one or bothof the 12^(th) and 13^(th) carbons, wherein the substituants areselected from the group consisting of hydroxy, oxo, halogen, amino,cyano, azido, a singly or multiply or unsubstituted C₁-C₂ carbon chain,cyclopropane, cyclopropene, and epoxy.
 63. The nematicidal compositionof claim 62 wherein the 12^(th) and 13^(th) carbons are substituted withan epoxy group.
 64. The nematicidal composition of claim 62 wherein the12^(th) carbon is substituted with a hydroxy group.
 65. The nematicidalcomposition of claim 62 wherein the 12^(th) carbon is substituted withan oxo group.